Abstract

The present study was undertaken to evaluate the osteogenic effect of Cervi cornu pantotrichum (CCP) on bone marrow stromal cells (BMSCs) which showed differentiation towards the osteoblastic phenotype. Cells were exposed to different culture medium (dexamethasone group, CCPE group and control group). The activities of osteoblastic markers such as alkaline phosphatase (ALP), bone gla protein (BGP) and bone morphogenetic protein-2 (BMP-2) were detected by Elisa. Cell proliferation was measured by MTT assay. The bone nodule formation was measured with alizarin red stain. Data anlysis showed that CCPE had significantly promoted proliferation of BMSCs in the first three days and stimulated the BMSCs differentiate into osteoblast. Calcium nodule was found after 18days. This results demonstrate that CCPE has a stimulatory effect on bone formation and maight be a useful remedy for the treatment of bone diseases such as osteoporosis.

Keywords

Introduction

Cervi cornu pantotrichum, the yong unossified antler of
male deer, has been one of the most prized health tonics
in traditional oriental medicine for over 2,000 years. As it
has a unique natural model of rapid and complete bone
regeneration, CCP has received increasing attention
because of its possible role in bone formation. Zhou et al.
showed that a polypeptide from deer velvet accelerates
fracture healing in laboratory mice by stimulating bone
and cartilage formation [1]. The growth factor of deer
antler enhanced the proliferation and differentiation of
human alveolar osteoblast cells. Chloroform extract of
deer antler inhibited osteoclast differentiation and bone
resorption [2]. Bone regeneration is promoted by
osteoconduction mechanisms; osteoinduction and
osteogenesis. Bone marrow stromal cells (BMSCs) have
long been recognized as the source of osteoprogenitor
cells [3]. However, whether Cervi cornu pantotrichum
aqueous extract is able to influence BMSCs has not been
reported. The current study is to determine the effect of
CCPE on the proliferation and differentiation of BMSCs.

Preparation of serum

Sixteen female SPF Sprague-Dawley (SD) rats weighing
200-220g were randomly divided into two groups. All the animals were purchased from Liaoning Changsheng
biotechnology co., LTD (China), and housed in a room at
temperature of 21-25, relative humidity of 50%-60%
and a 12h light/12h dark cycle. Animals had free access to
pellet food and tap water. Eight rats were given CCPE
solution (0.4g/kg) by gavage once a day for 8 days and
the others received water instead. Blood was aseptically
obtained from the abdominal aorta 2h after the last
administration. Serum was acquired by blood
centrifugation at 3000 rpm for 10 min and then filtered
twice with 0.22μm cellulose acetate membrane. The
bottled serum was calefied in 56℃ water for 30 min, and
then stored at -20℃ for use.

The cells (4×104) were seeded, in triplicate, in 96-well
plates. Cultured cells were exposed to different medium.

The α-MEM containing 10% serum of normal rat,
100U/mL penicillin-streptomycin was used for the control
group. The α-MEM containing 10% FBS, 10-8 mol/L
dexamethasone, 50μg/mL ascorbic acid and 10 mmol/L β-
Glycerophosphate was used as the positive group. The α-
MEM containing 10% serum containing CCPE, 100U/mL
penicillin-streptomycin was used for CCPE group. After
being cultured for 24h, 48h, 72h respectively, BMSCs
were supplemented with 10μL MTT(5g/L), and incubated
for another 4h. Then the supermatant was discarded by
aspiration and 100uL DMSO was added to dissolve the
dark blue crystal formazan thoroughly. The absorbance
was measured at 570 nm by multifunctional microplate
reader (TECAN Infinite M200, Austria).

Assay of cellar ALP, BGP, and BMP-2 Activities

The cells (2×104) were seeded in 24-well plates in three
copies, treated with the three medium which was said
above for 6d, 12d, 18d, washed twice with PBS, digested
with 0.25% trypsin, frozen and thawed three times, then
centrifuged at 3000 rpm for 10 min at 4. The ALP, BGP,
BMP-2 activities were assayed with commercial kits. The
absorbance was read at 450nm by multifunctional
microplate reader.

Alizarin red staining

BMSCs were seeded in 24-well plates at a density of
2×104 cells/well. After treatment with different medium
for 18days, cells were fixed with phosphate buffered
formalin and then stained with 40 mM alizarin red
solution for 30 min. After washing the wells with pure
water, the plates were photographed.

Statistical Methods

All of the data were expressed as mean values ± Standard
deviations. Statistical analysis was performed with oneway
analysis of variance (ANOVA) using SPSS 16.0
software. The arithmetic means, standard deviations (SD)
and 95% confidence intervals were calculated for each
experimental group.

Results and Discussion

Proliferation and differentiation of BMSCs

As seen in Fig.1 A, the number of the cells grown in 10%
serum containing CCPE was significantly higher than
those in control group and positive group at each time
point (p<0.05). This result showed that CCPE had such
functions as promoting proliferation of BMSCs. The
stromal cells isolated from bone marrow can be induced
by dexamethasone to differentiate into cells possessing
the characteristics of mature osteoblast [4]. Bone
formation required three essential steps such as osteoblast
differentiation, matrix maturation and matrix
mineralization [5] and was characterized by cellular
expression and synthesis of bone marker proteins for matrix maturation and by extracellular matrix
mineralization triggered by calcium deposition [6]. ALP
was the transporter protein binding with calcium, and it
could promote the cell maturation and calcification. BGP
was the necessary factor for bone calcification. ALP and
BGP were the markers in the differentiation in the early
stage and middle stage [7]. BMP-2 is well known as one
of the most powerful osteogenesis promoting proteins[8-11]. During osteoblast differentiation, BMP-2 was bound
with 2 types of serine / threonine kinase receptors-BMPR
Type I and Type II- initiated the BMP signaling pathway
subsequently[12-15]. Firstly, BMP gave the signal to
activate receptor-regulated Smads (R-Smad; Smadl,
Smad5, and Smad8), and regulated target genes such as
OPN, BGP, ALP, and Runx2. The research found that
ALP activities in both CCPE group and the
dexamethasone group were notably higher than that in the
control group (Fig.1B, p<0.05). The BGP activities in
dexamethasone group were higher than both CCPE group
and control group (Fig.1C, p<0.05). The BMP-2 activities
in CCPE group was significant higher than those in
dexamethasone group and control group (Fig.1D,
p<0.05).This study demonstrated that serum containing
CCPE not only enhanced proliferation of BMSCs but also
promoted the BMSCs to differentiate into osteoblast.
Further research will be necessary to assess the effect of
CCPE on bone regeneration and therapeutic potential in
bone formation in a model in vivo.

Figure 1: The effect of CCPE on cell proliferation in
BMSCs is measured by MTT assay at three different time
points. (n = 8). And cell viability data are expressed as
the optical density.

Figure 2: Mineralization of the BMSC?s after staining with Alizarin red

Alizarin red staining

After 18days, cultures were mineralized visually with
alizarin red staining as shown in Fig. 3. The BMSCs
cultured in both dexamethasone group and CCPE group
have differentiated into osteoblasts. They are stained with
dark alizarin red in extracellular and intracellular areas.
That was to say there were more mature osteoblasts in dexamethasone group and CCPE group than the cells in
control medium. The morphology of these cultures
strongly also suggests that cells have undergone an
osteoblastic differentiation.

As shown in Fig 1B, on 12th days, the osteoblasts treated
both with dexamethasone and CCPE have significantly
higher activity of ALP than that in the control group
(P<0.05). There is no significant difference between
osteoblasts in differentiation group and pilose anlter group (P>0.05).

The Fig.1 C shows that the BGP activities in both CCPE
group and dexamethasone group are significantly higher
than that in control group (P<0.05).

The Fig.1 D shows that on the 12 th, 18th days, the BMP-
2 activity in CCPE group is significantly higher than that
in differentiation group and the control group (P<0.05).

Acknowledgements

This work was supported by the National Science
Foundation of China (No.30973881).